RBC lysis for live cell flow cytometry

    1- Pellet the cells or use a minimal volume of suspended cells.
    2- Resuspend the cells in 1 ml of the lysing solution:

    Lysing solution

       Ammonium chloride (NH4Cl) 0.15M  8.29 g
       Potassium bicarbonate (KHCO3) 10mM  1 g
       EDTA  0.1mM     0.037 g
       H2O dist      1 l
       filtered with 0.45µm filter, stored at RT.


    3- Allow the red cell to lyse for 3-4 min at RT. Old RBCs may not lyse as well as the young ones.
    4- Add  6 ml of medium, mix.
    5- Layer at the bottom of the tube approx 0.5 ml of calf serum, by using a Pasteur pipette.
    6- Centrifuge the undisturbed layers for 5 min.
    7- Aspirate the supernatant, beginning with the medium+lysing solution.
    8- Add a suitable amount of complete medium for counting.